Bh3 peptides and methods of use thereof

ABSTRACT

The invention provides peptides and the nucleic acid sequences that encode them. The invention further provides therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of apoptosis associated disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/966,821, filedDec. 13, 2010, which is a continuation of U.S. Ser. No. 11/789,557,filed Apr. 24, 2007, now U.S. Pat. No. 7,868,133, which is acontinuation of U.S. Ser. No. 10/658,028, filed Sep. 9, 2003, nowabandoned, which claims priority to U.S. Ser. No. 60/409,488, filed Sep.9, 2002 and U.S. Ser. No. 60/495,036, filed Aug. 14, 2003, the contentsof all of which are incorporated herein by reference in theirentireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under NIH grantCA92625. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to methods and compositions for theregulation of apoptosis.

BACKGROUND OF THE INVENTION

Programmed cell death, referred to as apoptosis, plays an indispensablerole in the development and maintenance of tissue homeostasis within allmulticellular organisms (Raff, Nature 356: 397-400, 1992). Genetic andmolecular analysis from nematodes to humans has indicated that theapoptotic pathway of cellular suicide is highly conserved (Hengartnerand Horvitz, Cell 76: 1107-1114, 1994). In addition to being essentialfor normal development and maintenance, apoptosis is important in thedefense against viral infection and in preventing the emergence ofcancer.

Diverse intrinsic death signals emanating from multiple subcellularlocales all induce the release of cytochrome c from mitochondria toactivate Apaf-1 and result in effector caspase activation. Proteins inthe BCL-2 family are major regulators of the commitment to programmedcell death as well as executioners of death signals at themitochondrion.

Members of this family include both pro- and anti-apoptotic proteins andshare homology in up to four conserved regions termed BCL-2 homology(BH) 1-4 domains (Adams and Cory, 1998). The family can be divided intothree main sub-classes. The anti-apoptotic proteins, which include BCL-2and BCL-X_(L), are all “multidomain,” sharing homology throughout allfour BH domains. However, the pro-apoptotic proteins can be furthersubdivided and include multidomain proteins, such as BAX and BAK, whichpossess sequence homology in BH1-3 domains. The more distantly related“BH3-only” proteins are to date all pro-apoptotic and share sequencehomology within the amphipathic α-helical BH3 region, which is requiredfor their apoptotic function (Chittenden et al., 1995; O'Connor et al.,1998; Wang et al., 1996; Zha et al., 1997).

Multidomain pro-apoptotic proteins such as BAX and BAK upon receipt ofdeath signals participate in executing mitochondrial dysfunction. Inviable cells, these proteins exist as monomers. In response to a varietyof death stimuli, however, inactive BAX, which is located in the cytosolor loosely attached to membranes, inserts deeply into the outermitochondrial membrane as a homo-oligomerized multimer (Eskes et al.,2000; Gross et al., 1998; Wolter et al., 1997). Inactive BAK resides atthe mitochondrion where it also undergoes an allosteric conformnationalchange in response to death signals, which includes homo-oligomerization(Griffiths et al., 1999; Wei et al., 2000). Cells deficient in both BAXand BAK are resistant to a wide variety of death stimuli that emanatefrom multiple locations within the cell (Wei et al., 2001).

The BH3-only molecules constitute the third subset of this family andinclude BID, NOXA, PUMA, BIK, BIM and BAD (Kelekar and Thompson, 1998).These proteins share sequence homology only in the amphipathic α-helicalBH13 region which mutation analysis indicated is required inpro-apoptotic members for their death activity. Moreover, the BH3-onlyproteins require this domain to demonstrate binding to “multidomain”BCL-2 family members. Multiple binding assays, including yeasttwo-hybrid, co-immunoprecipitation from detergent solubilized celllysates and in-vitro pull down experiments indicate that individualBH3-only molecules display some selectivity for multidomain BCL-2members (Boyd et al., 1995; O'Connor et al., 1998; Oda et al., 2000;Wang et al., 1996; Yang et al., 1995). The BID protein bindspro-apoptotic BAX and BAK as well as anti-apoptotic BCL-2 and BCL-X_(L)(Wang et al., 1996; Wei et al., 2000). In contrast, BAD, NOXA and BIM asintact molecules display preferential binding to anti-apoptotic members(Boyd et al., 1995; O'Connor et al., 1998; Oda et al., 2000; Yang etal., 1995).

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the BH3 domain fromthe BCL-2 family of proteins alone can function as a specific deathligand. The peptides are refered to herein as BH3 peptides.

In one aspect the invention provides an isolated peptide having asequence of SEQ ID NO: 1, 2 or 10. The peptide induces BAKoligomerization and cytochrome c release from mitochondria. In anotheraspect the invention provides an isolated peptide having a sequence ofSEQ ID NOs: 3-9 or 11. The peptide binds BCL-2 or MCL-1. For example,SEQ ID NO: 1-5 binds BCL-2. Alternatively, SEQ ID NO: 6 and 7 bindMCL-1.

Also include in the invention is a chimeric peptide having a firstdomain and a second domain. The first domain having and amino acidsequence of SEQ ID NOs: 1-11. The second domain having a translocationsequence which facilitates transport across a biological membrane.Examples, of translocation sequence includes polyarginine.

In another aspect the invention includes a nucleic acid encoding any oneof the peptides of the invention.

Also included in the invention is a vector containing one or more of thenucleic acids described herein, and a cell containing the vectors ornucleic acids described herein.

The invention is also directed to host cells transformed with a vectorcomprising any of the nucleic acid molecules described above.

In another aspect, the invention includes a composition that includesthe peptides of the invention and a carrier or diluent.

In yet a further aspect the invention provides methods of treating acell proliferative disorder, e.g., cancer in a subject by administeringto a subject a BH3 peptide.

In another aspect the invention includes a method of inducing apoptosisin a cell by contacting said cell with SEQ ID NOs 1, 2 or 10 such thatapoptosis is induced. Alternatively, the invention provides a method ofsensitizing a cell to apoptosis by contacting said cell with acomposition comprising any of SEQ ID NOs: 3-7 or 11 such that as tosensitize the cell to apoptosis.

A further aspect the invention includes a method of screening for anapoptotic sensitizer compound by contacting mitochondria overexpressingBCL-2 with a BID-like BH3 peptide to form a BCL-2-peptide complex andcontacting the complex with a test compound. Cytochrome c release fromthe mitochondria is determined and compared to Cytochrome c release fromthe mitochondria not exposed to the compound. An increase of cytochromec release in the presence of the test compound compared to the absenceof the compound indicates the compound is an apoptotic sensitizercompound.

In another aspect, the invention relates to a transgenic animalcontaining a heterologous gene construct encoding a protein comprisingBCL-2 protein, or a cell isolated from this animal. The gene constructis ubiquitously expressed. Alternatively, the gene construct isconstitutively expressed. The gene constructs contain one or moreregulatory sequences, such as a promoter. For example, the geneconstruct is under the control of an inducible promoter. The transgenicanimal is useful for in vitro testing to determine the effect of a BCL-2antagonist.

In another aspect, the invention relates to a method of using cell linesisolated from a transgenic animal in an in vitro assay to determine theinhibition of a BCL-2 protein, inhibition of an anti-apoptotic BCL-2protein family member or determine the effects or antagonist thereof.

In another aspect, the invention relates to a transgenic non-humananimal containing a recombinant nucleic acid molecule stably integratedin its genome, where the recombinant nucleic acid molecule encodes aBCL-2 protein.

In a further aspect, the invention relates to a method for theproduction of a transgenic non-human animal, which includes introductionof a recombinant nucleic acid molecule into a germ cell, an embryoniccell, an egg cell or a cell derived therefrom, where the recombinantnucleic acid molecule encodes a BCL-2 protein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B. are bar charts showing BIDBH3, myrBID, and BIMBH3proteins and peptides induce cytochrome c release from mitochondria.

FIG. 2. is a photograph of an immunoblot showing BAX and BAK expressionin mitochondria isolated from mouse liver and FL5.12 cells.

FIG. 3. is a bar chart showing Cytochrome c release induced by BIMBH3and BIDBH3 is dependent on the presence of the multi-domainpro-apoptotic BAK.

FIG. 4A. is an immunoblot showing BAK oligomerization in Wt livermitochondria treated with 100 μM BIDBH3.

FIG. 4B. is a line graph showing peptide induced cytochrome C release.Wt liver mitochondria were treated as in (A) and cytochrome c releasemeasured by ELISA.

FIG. 4C. is two blots: upper panel is a blot showing BAK oligomerizationinduced by treatment if in mitochondria from FL5.12 cells with 100 μM ofthe indicated peptides. Markers 1, 2, 3, 4 correspond to size ofmonomer, dimer, trimer and tetramer; and lower panel is a blot showingBAX oligomerization in mitochondria from FL5.12 cells.

FIG. 5A. is a bar chart showing BCL-2 inhibits the release of cytochromec in mitochondria isolated from parental and BCL-2 over-expressingFL5.12 cells.

FIG. 5B. is a blot showing the oligomerization of BAK in mitochondriafrom parental and FL5.12-BCL-2 cells treated with 10 μM BIDBH3,incubated with cross-linking agent BMH, and SDS-PAGE and immunoblot forBAK.

FIG. 6 A. is a bar chart showing BADBH3 enables cytochrome c release byBIDBH3, BIMBH3 and myrBID.

FIG. 6B. is a graph showing BADBH3 enables cytochrome c release byBIDBH3 in a dose dependent fashion.

FIG. 6C. is a graph showing BADBH3 enables cytochrome c release byBIMDBH3 in a dose dependent fashion.

FIG. 6D. is a graph showing BADBH3 enables cytochrome c release bymyrBID in a dose dependent fashion.

FIG. 6E. is bar chart showing the dose response of BADBH3 and BIKBH3enabling myrBID-induced release of cytochrome c from mitochondria ofFL5.12-BCL-2 cells.

FIG. 7A. is a graph showing binding of BIDBH3 and BADBH3 binding toGST-BCL-2

FIG. 7B. is a graph showing displacement of BIDBH3 binding to GST-BCL-2by BADBH3

FIG. 7C. is a schematic model of the BID-Like domain.

FIG. 7D. is a is a schematic model of the BAD-Like domain.

FIG. 8. is a bar chart showing r8BADBH3 sensitizes Jurkat cells tor8BIDBH3 killing.

FIG. 9. is a schematic representation of a BH3-mimetic screeningstrategy.

FIG. 10. is a schematic showing the germ-line transmission of tet-Bcl-2allele.

FIG. 11. is a bar chart showing that the loss of BCL-2 expressioninduced by doxycycline treatment induces a dramatic, 1-2 log decrease inWBC and a remission of the leukemia

FIG. 12. are photographs of a Western Blot depicting the expression ofhBcl-2 in the spleen.

FIG. 13. is bar chart depicting the requirement of BCL-2 expression forleukemia cell survival.

FIG. 14. is bar chart depicting the requirement of IL-7 for leukemiacell growth in culture.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that peptidescomprising the BH3 domain from the BCL-2 family of proteins can functionas a specific death ligand. The peptide of the invention were derived inpart from the BH3 domain of BID, BIM, BAD, BIK, NOXA, and BCLXpolypeptides and initiate cell death either by activating pro-apoptoticmembers or by counteracting anti-apoptotic members, by displacing BH3domains from their pockets. The peptides which activate pro-apoptoticmembers are referred to herein as “BID-like BH3 peptides” (e.g., SEQ IDNO: 1, 2, and 10) whereas the peptides that counteract anti-apoptoticmembers, are refered to herein as “BAD-like BH3 peptide” (e.g., SEQ IDNO: 3-7 and 11). The BID-like and BAD-like peptide are summarized belowin Table 1 and are collectively refered to herein as BH3 peptides.Additionally, the invention provides methods and pharmaceuticalcompositions for treating pathophysiologies associated with apoptosis,e.g., cell proliferative disorders.

TABLE 1 Kd (nM), IC50 (nM), % Kd (nM) Amino Acid SEQ BCL-2 SD BID Dis-SD Helicity MCL-1 SD Sequence ID NO Binding +/- placement +/- (222 nM)Binding (+/-) BIDBH3 EDIIRNIARH 1 220 30 838  192 19.5 283 11 LAQVGDSMDRBIMBH3 MRPEIWIAQE 2 74 2 ND - 15.8 79 4 LRRIGDEFNA mBADBH3 LWAAQRYGRE 339 6 173   60 ND >2600 LRRMSDEFEG SFKGL BADBH3 NLWAAQRYGR 4 41 11 ND -23.9 ELRRMSDEFV DSFKK BIKBH3 MEGSDALALR 5 485 272 4920 1648 8.5LACIGDEMDV NOXAABH3 AELPPEFAAQ 6 >1000 - >30,000 - 5.1 752 334LRKIGDKVYC NOXABBH3 PADLKDECAQ 7 >1000 - >30,000 - 11.6 829 149LRRIGDKVNL BCLXBH3 VIPMAAVKQA 8 >1000 - >30,000 - 3.9 LREAGDEFELBIDBH3 mut EDIIRNIARH 9 >1000 - >30,000 - 16.4 >2600 AAQVGASMDR BID-likeXXXXXXIAXX 10 consensus LXXXGDXXXX BAD-like XXXXXXXXXX 11 consensusLXXXXDXXXX

BH3 Peptides

In one aspect, the invention provides a BH3 peptide. No particularlength is implied by the term “peptide”. In some embodiments, the BH3peptide is less than 195 amino acids in length, e.g., less than or equalto 150, 100, 75, 50, 35, 25 or 15 amino acid in length. For example aBH3 peptide includes the sequence of SEQ ID NO: 1-11. In variousembodiments, the BH3 peptide includes the amino acid sequence of SEQ IDNO: 1-2 or 10 where the peptide induces BAK oligomerization andcytochrome c mobilization (e.g., release of cytochrome c from themitochondria). By BAK oligomerization is meant that the BH3 peptideinduces the formation of BAK oligomers, e.g., dimers, trimers, etc. Theoligomers are hetero-oligomers. Alternatively, the oligomers arehomo-oligomers. In a further embodiment, the BH3 peptide stimulatesapoptosis, e.g., programmed cell death. Alternatively the BH3 peptidesincludes the amino acid sequence of SEQ ID NO: 3-5 or 11, where thepeptide binds BCL-2 or other anti-apoptotic members of the BCL-2 familyof proteins. Alternatively the BH3 peptides includes the amino acidsequence of SEQ ID NO: 6 or 7, where the peptide binds MCL-1 or otheranti-apoptotic members of the BCL-2 family of proteins. (See, Table 1).

Examples of BID-like BH3 peptides include a peptide which includes (inwhole or in part) the sequence NH₂-XXXXXXIAXXLXXXGDXXXX-COOH (SEQ IDNO:10). Examples of BAD-like BH3 peptides includes (in whole or in part)the sequence NH₂-XXXXXXXXXXLXXXXDXXXX-COOH (SEQ ID NO:11). As usedherein X may be any amino acid. Alternatively, the BID-like or BAD-likeBH3 peptides include at least 5, 6, 7, 8, 9, 15 or more amino acids ofSEQ ID NO: 10 or SEQ ID NO: 11)

The BH3 peptides can be polymers of L-amino acids, D-amino acids, or acombination of both. For example, in various embodiments, the peptidesare D retro-inverso peptides. The term “retro-inverso isomer” refers toan isomer of a linear peptide in which the direction of the sequence isreversed and the chirality of each amino acid residue is inverted. See,e.g., Jameson et al., Nature, 368, 744-746 (1994): Brady et al., Nature,368, 692-693 (1994). The net result of combining D-enantiomers andreverse synthesis is that the positions of carbonyl and amino groups ineach amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. Unless specifically statedotherwise, it is presumed that any given L-amino acid sequence of theinvention may be made into an D retro-inverso peptide by synthesizing areverse of the sequence for the corresponding native L-amino acidsequence.

Alternatively, the BH3 peptides are cyclic peptides. BH3 cyclic peptideare prepared by methods known in the art. For example, macrocyclizationis often accomplished by forming an amide bond between the peptide N-and C-termini, between a side chain and the N- or C-terminus [e.g., withK₃Fe(CN)₆ at pH 8.5](Samson et al., Endocrinology, 137: 5182-5185(1996)), or between two amino acid side chains. See, e.g., DeGrado, AdvProtein Chem, 39: 51-124 (1988).

Preparation of BH3 Peptide

BH3 peptides are easily prepared using modern cloning techniques, or maybe synthesized by solid state methods or by site-directed mutagenesis. ABH3 peptide may include dominant negative forms of a polypeptide. In oneembodiment, native BH3 peptides can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, BH3 polypeptides areproduced by recombinant DNA techniques. Alternative to recombinantexpression, BH3 peptides can be synthesized chemically using standardpeptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the BH3peptide is derived, or substantially free from chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of BH3 peptides inwhich the protein is separated from cellular components of the cellsfrom which it is isolated or recombinantly produced. In one embodiment,the language “substantially free of cellular material” includespreparations of BH3 peptides having less than about 30% (by dry weight)of non-BH3 peptide (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-BH3 peptide, stillmore preferably less than about 10% of non-BH3 peptide, and mostpreferably less than about 5% non-BH3 peptide. When the BH3 peptide orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of BH3 peptides in which the protein isseparated from chemical precursors or other chemicals that are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of BH3 peptides having less than about 30% (by dry weight)of chemical precursors or non-BH3 peptide chemicals, more preferablyless than about 20% chemical precursors or non-BH3 peptide chemicals,still more preferably less than about 10% chemical precursors or non-BH3peptide chemicals, and most preferably less than about 5% chemicalprecursors or non-BH3 peptide chemicals.

The term “biologically equivalent” is intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same apoptosis modulating effects, i.e., release ofcytocrome c or BAK oligomerization although not necessarily to the samedegree as the BH3 polypeptide deduced from sequences identified fromcDNA libraries of human, rat or mouse origin or produced fromrecombinant expression symptoms.

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to sequences asindicated above for identity comparisons. Conservative amino acidchanges satisfying this requirement are: R-K; E-D, Y-F, L-M; V-I, Q-H.

BH3 peptides can also include derivatives of BH3 peptides which areintended to include hybrid and modified forms of BH3 peptides includingfusion proteins and BH3 peptide fragments and hybrid and modified formsin which certain amino acids have been deleted or replaced andmodifications such as where one or more amino acids have been changed toa modified amino acid or unusual amino acid and modifications such asglycosylation so long as the hybrid or modified form retains thebiological activity of BH3 peptides. By retaining the biologicalactivity, it is meant that cell death is induced by the BH3 polypeptide,although not necessarily at the same level of potency as that of thenaturally-occurring BH3 polypeptide identified for human or mouse andthat can be produced, for example, recombinantly. The terms induced andstimulated are used interchangeably throughout the specification.

Preferred variants are those that have conservative amino acidsubstitutions made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a BH3polypeptide is replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of a BH3 coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedto identify mutants that retain activity.

Also included within the meaning of substantially homologous is any BH3peptide which may be isolated by virtue of cross-reactivity withantibodies to the BH3 peptide described herein or whose encodingnucleotide sequences including genomic DNA, mRNA or cDNA may be isolatedthrough hybridization with the complementary sequence of genomic orsubgenomic nucleotide sequences or cDNA of the BH3 peptides herein orfragments thereof.

Chimeric and Fusion Proteins

The invention also provides BH3 chimeric or fusion proteins. As usedherein, a BH3 or BID mutein “chimeric protein” or “fusion protein”comprises a BH3 or BID mutein polypeptide operatively linked to anon-BH3 polypeptide. An “BH3 peptide” refers to a polypeptide having anamino acid sequence corresponding to a BH3 peptide whereas a “non-BH3peptide refers to a polypeptide having an amino acid sequencecorresponding to a protein that is not substantially homologous to theBH3 peptide, e.g., a protein that is different from the BH3 peptide andthat is derived from the same or a different organism. Within a BH3peptide the BH3 peptide can correspond to all or a portion of a BH3peptide. In one embodiment, a BH3 peptide fusion protein comprises atleast one biologically active portion of a BH3 peptide. In anotherembodiment, a BH3 peptide fusion protein comprises at least twobiologically active portions of a BH3 peptide. Within the fusionprotein, the term “operatively linked” is intended to indicate that theBH3 peptide and the non-BH3 peptide are fused in-frame to each other.The non-BH3 peptide can be fused to the N-terminus or C-terminus of theBH3 peptide.

For example, in on aspect the invention provides a chimeric peptide thatinclude a first domain containing BH3 peptide operably linked to asecond domain containing a translocation sequence

A “translocation sequence” refers to any sequence of amino acids thatdirects a peptide in which it is present to a desired cellulardestination. For example the translocation sequence is polyarginine.Thus, the translocation sequence can direct or facilitate penetration ofthe peptide across a biological membrane, e.g., a phospholipid membrane,mitochondrial membrane, or nuclear membrane. For example thetranslocation sequence directs the peptide from outside the cell,through the plasma membrane, and into the cytoplasm or to a desiredlocation within the cell, e.g., the nucleus, the ribosome, themitochondria, the ER, a lysosome, or peroxisome. Alternatively, or inaddition, the translocation sequence can direct the peptide across aphysiological barrier such as the blood-brain barrier, the trans-mucosalbarrier, or the hematoencephalic, hematoretinal, gastrointestinal andpulmonary barriers.

Alternatively, a BH3 peptide fusion protein comprises a BH3 peptideoperably linked to the extracellular domain of a second protein. Suchfusion proteins can be further utilized in screening assays forcompounds that modulate BH3 peptide activity (such assays are describedin detail below).

In another embodiment, the fusion protein is a GST-BH3 peptide fusionprotein in which the BH3 peptide sequences are fused to the C-terminusof the GST (i.e., glutathione S-transferase) sequences. Such fusionproteins can facilitate the purification of recombinant BH3 peptide.

In another embodiment, the fusion protein is a BH3peptide-immunoglobulin fusion protein in which the BH3 peptide sequencescomprising one or more domains are fused to sequences derived from amember of the immunoglobulin protein family. The BH3peptide-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a BH3 peptide ligand and a BH3peptide on the surface of a cell, to thereby suppress BH3peptide-mediated signal transduction in vivo. In one nonlimitingexample, a contemplated BH3 peptide ligand of the invention is a VHLpolypeptide. The BH3 peptide-immunoglobulin fusion proteins can be usedto affect the bioavailability of a BH3 peptide cognate ligand.Inhibition of the BID α6 peptide ligand/BH3 peptide interaction may beuseful therapeutically for both the treatment of proliferativedisorders, as well as modulating (e.g., inducing or inhibiting) cellsurvival or apoptosis. For example, inhibition of the BH3 peptideligand/BH3 peptide can be used to various disorders as described herein.Moreover, the BH3 peptide-immunoglobulin fusion proteins of theinvention can be used as immunogens to produce anti-BH3 antibodies in asubject, to purify BH3 peptide ligands, and in screening assays toidentify molecules that inhibit the interaction of BH3 peptide with aBH3 peptide ligand.

In another embodiment, the fusion protein is a BH3 peptide-basic chargeddomain fusion protein in which the BH3 peptide sequences comprising oneor more domains are fused to a basic peptide domain. The BH3peptide-basic charged domain fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a BH3 peptide ligand and a BH3peptide in a cell, to thereby suppress BH3 peptide-mediated signaltransduction in vivo. Several examples of biologically active fusionproteins, comprising basic peptide domains, for direct delivery ofproteins into human patients in the context of protein therapy are knownin the art, including, but not limited to, the human immunodeficiencyvirus type 1 (HIV-1) TAT protein, HIV-1 Rev protein, DrosophilaAntennapedia or HIV-1 octaarginine protein. These basic peptide domainscan be arginine-rich. These transducing proteins have been shown to havea membrane permeability and a carrier function for the delivery ofproteins to the cytoplasm and nucleus of cells, both in vivo and invitro. These cells can be mammalian cells (i.e. human cells) (Suzuki etal., J Biol Chem 276: 5836-40, 2001 and Suzuki et al., J Biol Chem 277:2437-43, 2002).

A BH3 chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (Eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A BH3 peptide-encoding nucleic acid can be cloned intosuch an expression vector such that the fusion moiety is linked in-frameto the BH3 peptide.

BH3 Nucleic Acids

The present invention additionally relates to nucleic acids that encodeBH3 peptide. Nucleic acids encoding the BH3 peptides may be obtained byany method known in the art (e.g., by PCR amplification using syntheticprimers hybridizable to the 3′- and 5′-termini of the sequence and/or bycloning from a cDNA or genomic library using an oligonucleotide sequencespecific for the given gene sequence).

For recombinant expression of one or more BH3 peptides, the nucleic acidcontaining all or a portion of the nucleotide sequence encoding thepeptide may be inserted into an appropriate expression vector (i.e., avector that contains the necessary elements for the transcription andtranslation of the inserted peptide coding sequence). In someembodiments, the regulatory elements are heterologous (i.e., not thenative gene promoter). Alternately, the necessary transcriptional andtranslational signals may also be supplied by the native promoter forthe genes and/or their flanking regions.

A variety of host-vector systems may be utilized to express the peptidecoding sequence(s). These include, but are not limited to: (i) mammaliancell systems that are infected with vaccinia virus, adenovirus, and thelike; (ii) insect cell systems infected with baculovirus and the like;(iii) yeast containing yeast vectors or (iv) bacteria transformed withbacteriophage, DNA, plasmid DNA, or cosmid DNA. Depending upon thehost-vector system utilized, any one of a number of suitabletranscription and translation elements may be used.

Promoter/enhancer sequences within expression vectors may utilize plant,animal, insect, or fungus regulatory sequences, as provided in theinvention. For example, promoter/enhancer elements can be used fromyeast and other fungi (e.g., the GAL4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter). Alternatively, or in addition, they mayinclude animal transcriptional control regions, e.g., (i) the insulingene control region active within pancreatic β-cells (see, e.g.,Hanahan, et al., 1985. Nature 315: 115-122); (ii) the immunoglobulingene control region active within lymphoid cells (see, e.g., Grosschedl,et al., 1984. Cell 38: 647-658); (iii) the albumin gene control regionactive within liver (see, e.g., Pinckert, et al., 1987. Genes and Dev 1:268-276; (iv) the myelin basic protein gene control region active withinbrain oligodendrocyte cells (see, e.g., Readhead, et al., 1987. Cell 48:703-712); and (v) the gonadotropin-releasing hormone gene control regionactive within the hypothalamus (see, e.g., Mason, et al., 1986. Science234: 1372-1378), and the like.

Expression vectors or their derivatives include, e.g. human or animalviruses (e.g., vaccinia virus or adenovirus); insect viruses (e.g.,baculovirus); yeast vectors; bacteriophage vectors (e.g., lambda phage);plasmid vectors and cosmid vectors.

A host cell strain may be selected that modulates the expression ofinserted sequences of interest, or modifies or processes expressedpeptides encoded by the sequences in the specific manner desired. Inaddition, expression from certain promoters may be enhanced in thepresence of certain inducers in a selected host strain; thusfacilitating control of the expression of a genetically-engineeredpeptides. Moreover, different host cells possess characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation, andthe like) of expressed peptides. Appropriate cell lines or host systemsmay thus be chosen to ensure the desired modification and processing ofthe foreign peptide is achieved. For example, peptide expression withina bacterial system can be used to produce an unglycosylated corepeptide; whereas expression within mammalian cells ensures “native”glycosylation of a heterologous peptide.

Also included in the invention are derivatives, fragments, homologs,analogs and variants of BH3 peptides and nucleic acids encoding thesepeptides. For nucleic acids, derivatives, fragments, and analogsprovided herein are defined as sequences of at least 6 (contiguous)nucleic acids, and which have a length sufficient to allow for specifichybridization. For amino acids, derivatives, fragments, and analogsprovided herein are defined as sequences of at least 4 (contiguous)amino acids, a length sufficient to allow for specific recognition of anepitope.

The length of the fragments is less than the length of the correspondingfull-length nucleic acid or polypeptide from which the BH3 peptides s,or nucleic acid encoding same, is derived. Derivatives and analogs maybe full length or other than full length, if the derivative or analogcontains a modified nucleic acid or amino acid. Derivatives or analogsof the BH3 peptides include, e.g., molecules including regions that aresubstantially homologous to the peptides, in various embodiments, by atleast about 30%, 50%, 70%, 80%, or 95%, 98%, or even 99%, identity overan amino acid sequence of identical size or when compared to an alignedsequence in which the alignment is done by a computer homology programknown in the art. For example sequence identity can be measured usingsequence analysis software (Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705), with the defaultparameters therein.

In the case of polypeptide sequences, which are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thus,included in the invention are peptides having mutated sequences suchthat they remain homologous, e.g. in sequence, in function, and inantigenic character or other function, with a protein having thecorresponding parent sequence. Such mutations can, for example, bemutations involving conservative amino acid changes, e.g., changesbetween amino acids of broadly similar molecular properties. Forexample, interchanges within the aliphatic group alanine, valine,leucine and isoleucine can be considered as conservative. Sometimessubstitution of glycine for one of these can also be consideredconservative. Other conservative interchanges include those within thealiphatic group aspartate and glutamate; within the amide groupasparagine and glutamine; within the hydroxyl group serine andthreonine; within the aromatic group phenylalanine, tyrosine andtryptophan; within the basic group lysine, arginine and histidine; andwithin the sulfur-containing group methionine and cysteine. Sometimessubstitution within the group methionine and leucine can also beconsidered conservative. Preferred conservative substitution groups areaspartate-glutamate; asparagine-glutamine; valine-leucine-isoleucine;alanine-valine; phenylalanine-tyrosine; and lysine-arginine.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide, which is 50% identical to the referencepolypeptide over its entire length. Of course, other polypeptides willmeet the same criteria.

The invention also encompasses allelic variants of the disclosedpolynucleotides or peptides; that is, naturally-occurring alternativeforms of the isolated polynucleotide that also encode peptides that areidentical, homologous or related to that encoded by the polynucleotides.Alternatively, non-naturally occurring variants may be produced bymutagenesis techniques or by direct synthesis.

Species homologs of the disclosed polynucleotides and peptides are alsoprovided by the present invention. “Variant” refers to a polynucleotideor polypeptide differing from the polynucleotide or polypeptide of thepresent invention, but retaining essential properties thereof.Generally, variants are overall closely similar, and in many regions,identical to the polynucleotide or polypeptide of the present invention.The variants may contain alterations in the coding regions, non-codingregions, or both.

In some embodiments, altered sequences include insertions such that theoverall amino acid sequence is lengthened while the protein retainstrafficking properties. Additionally, altered sequences may includerandom or designed internal deletions that shorten the overall aminoacid sequence while the protein retains transport properties.

The altered sequences can additionally or alternatively be encoded bypolynucleotides that hybridize under stringent conditions with theappropriate strand of the naturally-occurring polynucleotide encoding apolypeptide or peptide from which the BH3 peptide is derived. Thevariant peptide can be tested for BH3 peptide-binding and modulation ofBH3 peptide-mediated activity using the herein described assays.‘Stringent conditions’ are sequence dependent and will be different indifferent circumstances. Generally, stringent conditions can be selectedto be about 5° C. lower than the thermal melting point (T_(M)) for thespecific sequence at a defined ionic strength and pH. The T_(M) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at pH 7 and the temperature is at least about 60°C. As other factors may affect the stringency of hybridization(including, among others, base composition and size of the complementarystrands), the presence of organic solvents and the extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one.

High stringency can include, e.g., Step 1: Filters containing DNA arepretreated for 8 hours to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 g/ml denatured salmon sperm DNA. Step 2: Filters arehybridized for 48 hours at 65° C. in the above prehybridization mixtureto which is added 100 mg/ml denatured salmon sperm DNA and 5-20×10⁶ cpmof ³²P-labeled probe. Step 3: Filters are washed for 1 hour at 37° C. ina solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes. Step 4:Filters are autoradiographed. Other conditions of high stringency thatmay be used are well known in the art. See, e.g., Ausubel et al.,(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley andSons, NY; and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORYMANUAL, Stockton Press, NY.

Moderate stringency conditions can include the following: Step 1:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/mldenatured salmon sperm DNA. Step 2: Filters are hybridized for 18-20hours at 55° C. in the same solution with 5-20×106 cpm ³²P-labeled probeadded. Step 3: Filters are washed at 37° C. for 1 hour in a solutioncontaining 2×SSC, 0.1% SDS, then washed twice for 30 minutes at 60° C.in a solution containing 1×SSC and 0.1% SDS. Step 4: Filters are blotteddry and exposed for autoradiography. Other conditions of moderatestringency that may be used are well-known in the art. See, e.g.,Ausubel et al., (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley and Sons, NY; and Kriegler, 1990, GENE TRANSFER ANDEXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

Low stringency can include: Step 1: Filters containing DNA arepretreated for 6 hours at 40° C. in a solution containing 35%formamnide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%Ficoll, 1% BSA, and 500 g/ml denatured salmon sperm DNA. Step 2: Filtersare hybridized for 18-20 hours at 40° C. in the same solution with theaddition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm ³²P-labeled probe.Step 3: Filters are washed for 1.5 hours at 55° C. in a solutioncontaining 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. Thewash solution is replaced with fresh solution and incubated anadditional 1.5 hours at 60° C. Step 4: Filters are blotted dry andexposed for autoradiography. If necessary, filters are washed for athird time at 65-68° C. and reexposed to film. Other conditions of lowstringency that may be used are well known in the art (e.g., as employedfor cross-species hybridizations). See, e.g., Ausubel et al., (eds.),1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, NY;and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY.

BH3 Antibodies

Also included in the invention are antibodies to BH3 peptides orfragments thereof. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin (Ig) molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen.Such antibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, F_(ab), F_(ab′), and F_((ab)′2) fragments, andan F_(ab) expression library. In general, an antibody molecule obtainedfrom humans relates to any of the classes IgG, IgM, IgA, IgE and IgD,which differ from one another by the nature of the heavy chain presentin the molecule. Certain classes have subclasses as well, such as IgG₁,IgG₂, and others. Furthermore, in humans, the light chain may be a kappachain or a lambda chain. Reference herein to antibodies includes areference to all such classes, subclasses and types of human antibodyspecies.

An isolated BH3-related protein of the invention may be intended toserve as an antigen, or a portion or fragment thereof, and additionallycan be used as an immunogen to generate antibodies thatimmunospecifically bind the antigen, using standard techniques forpolyclonal and monoclonal antibody preparation. The full-length proteincan be used or, alternatively, the invention provides antigenic peptidefragments of the antigen for use as immunogens. An antigenic peptidefragment comprises at least 6 amino acid residues of the amino acidsequence of the full length protein, or amino acid sequences as shown inSEQ ID NOs:1-7, and encompasses an epitope thereof such that an antibodyraised against the peptide forms a specific immune complex with the fulllength protein or with any fragment that contains the epitope. Byepitope reference is made to an antigenic determinant of a polypeptide.Typically, epitopes contain hydrophilic amino acids such that theparticular region of the polypeptide is located on its surface andlikely to be exposed in an aqueous based milieu. Preferably, theantigenic peptide comprises at least 3 amino acid residues in a spatialconformation which is unique to the epitope. Generally, the antigenicpeptide comprises at least 5 amino acid residues, or at least 10 aminoacid residues, or at least 15 amino acid residues, or at least 20 aminoacid residues, or at least 30 amino acid residues. Furthermore,antibodies to a BH3 peptide or fragments thereof can also be raisedagainst oligopeptides that include a conserved region such as the α6helix domain of BID identified herein.

In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a region of BH3 that is locatedon the surface of the protein, e.g., a hydrophilic region. Ahydrophobicity analysis of the human BH3 sequence will indicate whichregions of a BH3 are particularly hydrophilic and, therefore, are likelyto encode surface residues useful for targeting antibody production. Asa means for targeting antibody production, hydropathy plots showingregions of hydrophilicity and hydrophobicity may be generated by anymethod well known in the art, including, for example, the Kyte Doolittleor the Hopp Woods methods, either with or without Fouriertransformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci.USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142,each of which is incorporated herein by reference in its entirety.Antibodies that are specific for one or more domains within an antigenicprotein, or derivatives, fragments, analogs or homologs thereof, arealso provided herein.

A protein of the invention, or a derivative, fragment, analog, homologor ortholog thereof, may be utilized as an immunogen in the generationof antibodies that immunospecifically bind these protein components.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof (see, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference). Some of theseantibodies are discussed below.

Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byone or more injections with the native protein, a synthetic variantthereof, or a derivative of the foregoing. An appropriate immunogenicpreparation can contain, for example, the naturally occurringimmunogenic protein, a chemically synthesized polypeptide representingthe immunogenic protein, or a recombinantly expressed immunogenicprotein. Furthermore, the protein may be conjugated to a second proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. The preparation can further include an adjuvant. Variousadjuvants used to increase the immunological response include, but arenot limited to, Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,etc.), adjuvants usable in humans such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory agents. Additionalexamples of adjuvants which can be employed include MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate) and CpGdinucleotide motifs (Krieg, A. M. Biochim Biophys Acta 1489(1):107-16,1999).

The polyclonal antibody molecules directed against the immunogenicprotein can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

Monoclonal Antibodies

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having ahigh degree of specificity and a high binding affinity for the targetantigen are isolated.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include, for example, Dulbecco'sModified Eagle's Medium and RPMI-1640 medium. Alternatively, thehybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

Humanized Antibodies

The antibodies directed against the protein antigens of the inventioncan further comprise humanized antibodies or human antibodies. Theseantibodies are suitable for administration to humans without engenderingan immune response by the human against the administered immunoglobulin.Humanized forms of antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) that areprincipally comprised of the sequence of a human immunoglobulin, andcontain minimal sequence derived from a non-human immunoglobulin.Humanization can be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science. 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539.) In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

Human Antibodies

Fully human antibodies relate to antibody molecules in which essentiallythe entire sequences of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies”, or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) andthe EBV hybridoma technique to produce human monoclonal antibodies (seeCole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized inthe practice of the present invention and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). Similarly, human antibodies can be made by introducing humaninununoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10, 779-783 (1992)): Lonberg et al. (Nature 368 856-859(1994)): Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (NatureBiotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14,826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93(1995)).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a methodincluding deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker, andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

F_(ab) Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to an antigenic protein of theinvention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods canbe adapted for the construction of F, expression libraries (see e.g.,Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effectiveidentification of monoclonal F_(ab) fragments with the desiredspecificity for a protein or derivatives, fragments, analogs or homologsthereof. Antibody fragments that contain the idiotypes to a proteinantigen may be produced by techniques known in the art including, butnot limited to: (i) an F_((ab)2) fragment produced by pepsin digestionof an antibody molecule; (ii) an F, fragment generated by reducing thedisulfide bridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragmentgenerated by the treatment of the antibody molecule with papain and areducing agent and (iv) F_(v) fragments.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is foran antigenic protein of the invention. The second binding target is anyother antigen, and advantageously is a cell-surface protein or receptoror receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., 1991 EMBO J.,10:3655-3659.

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full-length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See. Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein (BID or BID α6).

Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies can be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Effector Function Engineering

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins. Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody can be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is in turnconjugated to a cytotoxic agent.

Methods of Modulating Apoptosis

Also included in the invention are methods of inducing apoptosis orsensitizing a cell to apoptosis. By “inducing apoptosis” is meant thatthat the program cell death is initiated. Apoptosis is measured bymethods known in the art, for example apoptosis is measured by annexin Vstaining.

In one aspect apoptosis is induced in subject in need thereof byadministering a BH3 peptide or BH3 chimeric peptide in an amountsufficient to induce apoptosis. The subject can be e.g., any mammal,e.g., a human, a primate, mouse, rat, dog, cat, cow, horse, pig. Invarious aspects the subject is susceptible to cancer or an autoimmunedisorder.

A BH3 peptide or BH3 chimeric peptide is administered with ananti-angiogenic compound. Examples of an anti-angiogenic compoundinclude, but are not limited to, a tyrosine kinase inhibitor, anepidermal-derived growth factor inhibitor, a fibroblast-derived growthfactor inhibitor, a platelet-derived growth factor inhibitor, a matrixmetalloprotease (MMP) inhibitor, an integrin blocker, interferon alpha,interferon-inducible protein 10, interleukin-12, pentosan polysulfate, acyclooxygenase inhibitor, a nonsteroidal anti-inflammatory (NSAID), acyclooxygenase-2 inhibitor, carboxyamidotriazole, tetrahydrocortizol,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, endostatin, troponin-1, an antibody to VEGF,platelet factor 4 or thrombospondin.

The BH3 peptide or BH3 chimeric peptide is further administered with anchemotherapeutic compound. Examples of chemotherapeutic compoundsinclude, but are not limited to, paclitaxel, Taxol, lovastatin,minosine, tamoxifen, gemcitabine, 5-fluorouracil (5-FU), methotrexate(MTX), docetaxel, vincristin, vinblastin, nocodazole, teniposide,etoposide, adriamycin, epothilone, navelbine, camptothecin,daunonibicin, dactinomycin, mitoxantrone, amsacrine, epirubicin oridarubicin.

Alternatively, the BH3 peptide or BH3 chimeric peptide is furtheradministered with an antibody, such as polyclonal, monoclonal,humanized, human, bispecific, heteroconjugate, immunoconjugate,chimeric, single chain, F_(ab), F_(ab′) and F_((ab′)2) fragments, and anF_(ab) expression library as described above.

In another aspect, apoptosis is induced in a cell by contacting a cellwith a BH3 peptide or BH3 chimeric peptide in an amount sufficient toinduce apoptosis. Alternatively, a cell is sensitized to apoptosis bycontacting a cell with a BH3 peptide or BH3 chimeric peptide in anamount sufficient to sensitize the cell to apoptosis. The cellpopulation that is exposed to, i.e., contacted with, the BH3 peptide orBH3 chimeric peptide can be any number of cells, i.e., one or morecells, and can be provided in vitro, in vivo, or ex vivo.

Some disease conditions are related to the development of a defectivedown-regulation of apoptosis in the affected cells. For example,neoplasias result, at least in part, from an apoptosis-resistant statein which cell proliferation signals inappropriately exceed cell deathsignals. Furthermore, some DNA viruses such as Epstein-Barr virus.African swine fever virus and adenovirus, parasitize the host cellularmachinery to drive their own replication. At the same time, theymodulate apoptosis to repress cell death and allow the target cell toreproduce the virus. Moreover, certain disease conditions such aslymphoproliferative conditions, cancer including drug resistant cancer,arthritis, inflammation, autoimmune diseases and the like may resultfrom a down regulation of cell death regulation. In such diseaseconditions, it would be desirable to promote apoptotic mechanisms.

Methods of Screening for Apoptotic Modulating Compounds

The invention further provides a method of screening for compound thatmodulate apoptosis. i.e., activators or sensitizers.

In various methods, a apoptotic sensitizer compound is identified bycontacting a mitochondrion overexpressing an anti-apoptotic protein,e.g. BCL-2 or BCL-X_(L) with a BID-like BH3 peptide to form aprotein-peptide complex. The complex is contacted with a candidatecompound, and cytochrome c release is determined and compared to theamount of cytochrome c release in the test population to a controlpopulation that has or has not been exposed to the compound An increasein cytochrome c release presence of the compound as compared to theabsence of the compound indicates the compound is an apoptoticsensitizer.

The invention also includes an apoptosis sensitizer identified accordingto this screening method, and a pharmaceutical composition whichincludes the apoptosis modulator.

Pharmaceutical Compositions

The compounds, e.g., BH3 peptides BH3 chimeric peptides, nucleic acidsencoding BH3 peptides, and BH3 and BH3 antibodies (also referred toherein as “active compounds”) of the invention, and derivatives,fragments, analogs and homologs thereof, can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, or protein,and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, finger's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a BH3 peptide or BH3 peptide encoding nucleic acid) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated fully herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Transgenic Animals.

In another aspect, the present invention includes transgenic animalshaving a heterologous (or exogenous) gene construct or transgeneencoding a BCL-2 polypeptide. (e.g., the tet-BCL-2 allele)

The preparation of a transgenic mammal includes introducing a nucleicacid construct that expresses a nucleic acid encoding a BCL-2polypeptide into an undifferentiated cell type, e.g., an embryonic stein(ES) cell. The ES cell is injected into a mammalian embryo, where itintegrates into the developing embryo. The embryo is implanted into afoster mother for the duration of gestation.

Embryonic stem cells are typically selected for their ability tointegrate into and become part of the germ line of a developing embryoso as to create germ line transmission of the heterologous geneconstruct. Thus, any ES cell line that has this capability is suitablefor use herein. One mouse strain that is typically used for productionof ES cells is the 129J strain. A preferred ES cell line is murine cellline D3 (American Type Culture Collection catalog no. CRL 1934). Morepreferably, the cell line is RW4. The cells are cultured and preparedfor DNA insertion using methods well known in the art, such as those setforth by Robertson (Robertson, In: Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed., IRL Press,Washington, D.C., 1987.). Insertion of the nucleic acid construct intothe ES cells can be accomplished using a variety of methods well knownin the art including for example, electroporation, microinjection, andcalcium phosphate treatment.

The term “transgene” is used herein to describe genetic material thathas been or is about to be artificially inserted into the genome of amammalian cell, particularly a mammalian cell of a living animal. Thetransgene is used to transform a cell, meaning that a permanent ortransient genetic change, preferably a permanent genetic change, isinduced in a cell following incorporation of an heterologous nucleicacid, such as DNA. A permanent genetic change is generally achieved byintroduction of the DNA into the genome of the cell. Vectors for stableintegration include plasmids, retroviruses and other animal viruses,YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs,goats, horses, etc., and particularly rodents, e.g., rats, mice, etc.Preferably, the transgenic animals are mice.

Transgenic animals comprise an heterologous nucleic acid sequencepresent as an extrachromosomal element or stably integrated in all or aportion of its cells, especially in germ cells. Unless otherwiseindicated, it will be assumed that a transgenic animal comprises stablechanges to the germline sequence. During the initial construction of theanimal, “chimeras” or “chimeric animals” are generated, in which only asubset of cells have the altered genome. Chimeras are primarily used forbreeding purposes in order to generate the desired transgenic animal.Animals having a heterozygous alteration are generated by breeding ofchimeras. Male and female heterozygotes are typically bred to generatehomozygous animals.

The heterologous gene is usually either from a different species thanthe animal host, or is otherwise altered in its coding or non-codingsequence. The introduced gene may be a wild-type gene, naturallyoccurring polymorphism, or a genetically manipulated sequence, forexample having deletions, substitutions or insertions in the coding ornon-coding regions. Where the introduced gene is a coding sequence, itis usually operably linked to a promoter, which may be constitutive orinducible, and other regulatory sequences required for expression in thehost animal. By “operably linked” is meant that a DNA sequence and aregulatory sequence(s) are connected in such a way as to permit geneexpression when the appropriate molecules, e.g., transcriptionalactivator proteins, are bound to the regulatory sequence(s). Thetransgenic animals of the invention can comprise other geneticalterations in addition to the presence of the heterologous gene. Forexample, the host's genome may be altered to affect the function ofendogenous genes comprising BH-3 domains (e.g., endogenous BID, BIM,BAD, BIK, NOXA and/or BCLX genes), contain marker genes, or othergenetic alterations. Construction of a BCL-2 transgenic mouse isillustrated in FIG. 10 where 16 chimeras were bred to B6 mice to produce45 positive F1 offspring.

Knockouts and Knockins

Although not necessary to the operability of the invention, thetransgenic animals described herein may comprise alterations toendogenous genes comprising BCL-2 in addition to the genetic alterationsdescribed above. For example, the host animals may be either “knockouts”and/or “knockins” for a target gene(s) as is consistent with the goalsof the invention (e.g., the host animal's endogenous BCL-2 gene may be“knocked out” and/or the BCL-2 gene may be “knocked in”). Knockouts havea partial or complete loss of function in one or both alleles of anendogenous gene comprising BCL-2 genes of interest. Knockins have anintroduced transgene with altered genetic sequence and/or function fromthe endogenous BCL-2 gene. The two may be combined, for example, suchthat the naturally occurring gene is disabled, and an altered formintroduced. For example, it may be desirable to knockout the hostanimal's endogenous gene comprising BCL-2, while introducing anexogenous gene comprising BCL-2.

In a knockout, preferably the target gene expression is undetectable orinsignificant. For example, a knock-out of a gene comprising BCL-2 meansthat function of the BCL-2 gene has been substantially decreased so thatexpression is not detectable or only present at insignificant levels.This may be achieved by a variety of mechanisms, including introductionof a disruption of the coding sequence, e.g., insertion of one or morestop codons, insertion of a DNA fragment, etc., deletion of codingsequence, substitution of stop codons for coding sequence, etc. In somecases the exogenous transgene sequences are ultimately deleted from thegenome, leaving a net change to the native sequence. Differentapproaches may be used to achieve the “knock-out”. A chromosomaldeletion of all or part of the native gene may be induced, includingdeletions of the non-coding regions, particularly the promoter region,3′ regulatory sequences, enhancers, or deletions of gene that activateexpression of BCL-2 genes. A functional knock-out may also be achievedby the introduction of an anti-sense construct that blocks expression ofthe native genes (See, e.g., Li and Cohen (1996) Cell 85:319-329).“Knock-outs” also include conditional knock-outs, for example wherealteration of the target gene occurs upon exposure of the animal to asubstance that promotes target gene alteration, introduction of anenzyme that promotes recombination at the target gene site (e.g. Cre inthe Cre-lox system), or other method for directing the target genealteration post-natally.

A “knockin” of a target gene means an alteration in a host cell genomethat results in altered expression or function of a native target gene.Increased (including ectopic) or decreased expression may be achieved byintroduction of an additional copy of the target gene, or by operativelyinserting a regulatory sequence that provides for enhanced expression ofan endogenous copy of the target gene. These changes may be constitutiveor conditional, i.e. dependent on the presence of an activator orrepresser. The use of knockin technology may be combined with productionof exogenous sequences to produce the transgenic animals of theinvention.

The heterologous gene construct includes a nucleic acid encoding aprotein comprising BCL-2 proteins. The heterologous gene construct canalso encode for various selection markers and enhancer elements.

A selection marker can be any nucleic acid sequence that is detectableand/or assayable. Examples of selection markers include positiveselection markers and negative selection markers. Positive selectionmarkers include drug resistance genes; e.g., neomycin resistance genesor hygromycin resistance genes, or beta-galactosidase genes. Negativeselection markers, e.g., thymidine kinase gene, diphtheria toxin geneand ganciclovir are useful in the heterologous gene construct in orderto eliminate embryonic stem (ES) cells that do not undergo homologousrecombination. The selection marker gene is usually operably linked toits own promoter or to another strong promoter from any source that willbe active or can easily be activated in the cell into which it isinserted; however, the marker gene need not have its own promoterattached as it may be transcribed using the promoter of the BCL-2containing gene to be suppressed. In addition, the marker gene willnormally have a polyA sequence attached to the 3′ end of the gene; thissequence serves to terminate transcription of the gene.

“Enhancer elements” include nucleic acid sequences that are bound bypolypeptides associated with transcription, and are usually in cis withthe nucleic acid encoding a light-generating fusion protein. Examples ofenhancer elements include cyclic AMP response elements (CRE), serumresponse elements (SRE), nuclear factor B (NF-κB), activator protein 1(AP-1), serum response factor (SRF), and p53 binding sites. Theseenhancer elements may further include a TATA box.

The heterologous gene construct may be constituitively expressed in thetransgenic mammal. The gene construct may expressed in specific tissues,e.g., the construct is under the control of a tissue-specific promoter.

The invention includes a transgenic mouse containing a heterologous geneconstruct encoding a BCL-2 protein. The gene construct is under thecontrol of a conditional promoter Activation of the promoter by atransgenic trans-activator protein results in increased expression ofthe gene construct encoding the BCL-2 protein. Inactivation of thetransactivator is achieved by the interaction of a selectedbiocompatible entity, or parts of the entity, with the transactivatorelements. This results in a decrease in expression of the BCL-2transgene. If the activation occurs only in a part of the animal, onlycells in that part will express the BCL-2 proteins.

Transgenic Cell Lines

The invention also includes cell lines derived from the transgenicanimals described above. An example cell line is derived from the bonemarrow of a triply transgenic (E-mu myc+/tet-Bcl-2+/MMTVtTA+) leukemicmouse. The cell line is dependent on IL-7 for division. When treatedwith doxycycline, which turns off BCL-2, all the cells die,demonstrating the BCL-2 dependence of the cell line. The invention alsoincludes a method of using the cell line in an in vitro assay todetermine the inhibition of BCL-2 by a peptide or peptidomimeticcomprising a BH3 domain (i.e. BCL-2 inhibition) or determine the effectsof a test compound (i.e. BH3 agonist).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1 General Methods Peptide Stocks

Peptides were synthesized by Tufts University Core Facility, purified byHPLC, and identity confirmed by mass spectroscopy. Stock solutions were10-20 mM DMSO.

Isolation of Mitochondria

Mouse liver mitochondria were isolated from age-matched wt or Bak −/−mice. Livers were diced, subjected to Dounce rotary Teflon pestledisruption, then homogenized using a Kinematica Polytron homognizer.Following suspension in isolation buffer (250 mM sucrose, 10 mM Tris-HClpH 7.4, 0.1 mM EGTA), mitochondria were isolated by differentialcentrifugation steps, followed by two washes in isolation buffer.Mitochondria from FL5.12 cells were isolated by cell disruption followedby differential centrifugation and washing as above. Cell disruption wasperformed either by a Kinematika Polytron homogenizer or by acombination of Dounce homogenization followed by 6-10 expulsions througha 27 gauge needle.

Cytochrome c Release

Mitochondria at a protein concentration of 0.5 mg/ml were treated atroom temperature in experimental buffer (125 mM KCl, 10 mM Tris-MOPS pH7.4, 5 mM glutamate, 2.5 mM malate, 1 mM KPO₄, 10 □M EGTA-Tris pH 7.4).Percent release was quantitated using a colorimetric ELISA (MCTC0, R&DSystems). In all experiments, treatments with DMSO were used as acontrol for solvent activity.

BMH Cross-Linking

1,6-Bismaleimidohexane was obtained from Pierce (#22330). A 10 mM stocksolution in DMSO was added to treated mitochondrial suspensions at a1:11 dilution. Cross-linking took place for 30 minutes at roomtemperature, followed by centrifugation to pellet mitochondria. Pelletswere dissolved in NuPAGE loading buffer (Invitrogen).

Binding Assays

To determine K_(d) for peptide binding to BCL-2, a GST-BCL-2 fusionprotein lacking the C-terminal transmembrane domain was utilized.Peptides were synthesized with a fluorescein amino-terminus using an AHAlinker. Peptides at 25 nM were mixed with titrations of GST-BCL-2 inbinding buffer (140 mM NaCL, 10 mM Tris, pH 7.4) at 37° C. An increasein fluorescence polarization measured on a Perkin-Elmer LS 50Bluminescence spectrophotometer was quantitated to calculate binding. Anon-linear fit to a sigmoidal dose-response curve utilized the programOrigin 6.0 to determine K_(d). For quantitative BIDBH3 displacementassays, 25 nM fluoresceinated BIDBH3 was mixed with 1 μM GST-BCL-2 inbinding buffer. Increasing amounts of unlabelled BH3 peptides weretitrated in, with loss of fluorescence polarization as a measurement ofdisplacement of BIDBH3. Data were fitted to a sigmoidal curve as above,and IC50 determined.

GST-BCL-2 Production

GST-BCL-2δC21 fusion proteins were induced in BL21 DE3 by 0.1 mM IPTG.The bacterial pellets were resuspended in lysis buffer (1 mg/mllysozyme/l % Triton X-100/0.1 mg/ml PMSF/2 μg/ml aprotinin/2 μg/mlleupeptine/1 μg/ml pepstatin A in PBS) and sonicated. Aftercentrifugation at 20,000×g for 20 min, the supernatant was applied toglutathione-agarose beads (Sigma). The beads were washed with PBS andtreated with 50 mM glutathione/50 mM TrisHCl, pH8.0 to elute protein.Eluate was dialyzed against binding buffer and concentrated using Amiconcentrifugal concentrating devices.

Circular Dichroism

Circular dichroism (CD) spectra were obtained on a Jasco J-710spectropolarimeter at 20° C. using the following standard measurementparameters: wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20nm/sec; accumulations, 10; response, 1 sec; band width, 1 nm; pathlength, 0.1 cm. Stock solutions of peptide were dissolved in deionizedwater and concentrations determined by amino acid analysis. Samples werethen diluted in 50 mM potassium phosphate pH 7 to a calculated finalconcentration of 50 μM. The CD spectrum of each sample was measured intriplicate and a background spectrum of diluent alone was subtracted.For comparison, the subtracted CD spectra were normalized to 35 μM basedon repeat peptide concentration determination by amino acid analysis ofthe diluted peptide solutions. The α-helical content of each peptide wascalculated by dividing the mean residue ellipticity [q]222obs by thereported [q]222obs for a model helical decapeptide (Yang et al., 1986).

Immunoblot Analysis

Antibodies used for immunoblot analysis included anti-cytochrome c(75981A, Pharmingen), anti-BAK (Upstate Biotechnology), and anti-BAX(N-20, Santa Cruz). Antibody detection was accomplished using enhancedchemiluminescence (Western Lightning, Perkin-Elmer).

Jurkat Cell Death

Jurkat cells were grown in RPMI 1640, 10% fetal bovine serum, 100 u/mlpenicillin, 100 μg/ml/strep, 2 mM glutamine, 50 μM β-mercaptoethanol.Cells were treated with peptide for 5 hours followed by staining withfluorescently-tagged Annexin V according to manufacturer's protocol (BDBiosciences 556547). Death was quantitated by FACS followed by analysisusing FlowJo software (Tree Star. Inc.)

Example 2 BH3 Peptides from BID and BIM, but not all BH3-Only MembersRelease Cytochrome C Similar to Myristoylated Bid

Recombinant p15tBID and even more efficiently the p7/myrp15,myristoylated BID complex (myrBID), initiate BAK oligomerization andcytochrome c release in a mitochondrial in-vitro system that appears torecapitulate the mitochondrial pathway of apoptosis in-vivo. Since thepro-apoptotic activity of BID in-vitro and in-vivo requires an intactBH3 domain, we tested the ability of peptides derived from this BH3domain to initiate this activity. A 20 mer of BIDBH3 (aa 80-99) at 10 μM(Table 1) proved capable of initiating cytochrome c release, as didmyrBID (FIG. 1A), the activity of other BH3 domain peptides werecompared. While BIMBH3 (Table 1) demonstrated cytochrome c release,peptides derived from other BH3-only members BAD, BIK, and NOXA(Table 1) even at 100 μM did not display this activity (FIG. 1B). Apeptide derived from the BH3 domain of anti-apoptotic Bcl-X_(L) did notcause cytochrome c release (FIG. 1B). Circular dichroism studiesindicate that while the relative α-helical content of these peptidesvaries, the percent α-helicity does not solely dictate the activity ofthe peptides. While NOXAABH3 and BCL-X_(L)BH3 have relatively lowα-helical content, BADBH3 demonstrates the highest α-helical content andis still inactive in this assay (Table 1). Likewise, a peptide derivedfrom the BH3 domain of BID, but containing substitutions (L90A, D95A) attwo residues highly conserved throughout the family, retainedα-helicity, but did not cause cytochrome c release (Table 1. FIG. 1B).

Example 3 BAK is Required for BH3 Peptide-Induced Cytochrome C Release

To test whether BH3 peptides work through an established mitochondrialpathway of apoptosis, we examined whether they required the“multidomain” member BAK to be present. One hallmark of cytochrome crelease by native tBID is that it requires the presence of multidomainBAK or BAX in intact cells or BAK on purified mitochondria (Wei et al.,2000; Wei et al., 2001). Immunoblots of mitochondria isolated from theliver of Bak −/− mice confirmed that neither “multi-domain”pro-apoptotic BAX nor BAK was present (FIG. 2). Moreover, there is nocompensatory alteration in the levels of anti-apoptotic BCL-2 members inthe absence of BAX and/or BAK (not shown). Comparison of 100 μM BIMBH3or BIDBH3 peptide on Bak +/+vs. −/− mitochondria indicated that BAK isrequired for the release of cytochrome c (FIG. 3). This requirement forBAK argues that these α-helical BH3 peptides function through thegenetic pathway of mitochondrial apoptosis rather than by an autonomouspermeabilization of membranes which non-specifically damagesmitochondria.

Example 4 Peptides that Induce Cytochrome C Release Induce BakOligomerization

Previous work demonstrated that the translocation of tBID to themitochondrion results in allosteric conformational activation of BAK,which includes its homo-oligomerization followed by the release ofcytochrome c (Wei et al., 2000). It was found that the BIDBH3 peptide(like tBID or the p7/myr15BID complex) induces BAK oligomerization asdetected with the cross-linker, BMH (FIG. 4A). Moreover, there istemporal relationship between BAK oligomerization and the release ofcytochrome c induced by BIDBH3 peptide (FIG. 4A,B). While BIMBH3 alsoinduces BAK oligomerization, BADBH3 peptide, which lacks the ability tocause cytochrome c release, is unable to induce BAK oligomerization(FIG. 4C). In was also found that BIMBH3 and BIDBH3, but not BADBH3,could also induce oligomerization of BAX in mitochondria isolated fromcultured FL5.12 cells, which contain both BAX and BAK (FIG. 2, 4C). Notethat while BIDBH3 induces more prominent cross-linking of BAK than doesBIMBH3, BIMBH3 induces more prominent cross-linking of BAX than doesBIDBH3. A mutant BID peptide BIDBH3mut (L90A, D95A) was tested and itlacked the ability to induce either cytochrome c release (FIG. 1B) orBAX, BAK oligomerization (FIG. 4C). These results indicate that BIDBH3and BIMBH3 peptides, like intact tBID protein are capable of inducing anallosteric change in mitochondrial-resident BAK or BAX, which includestheir homo-oligomerization and subsequent release of cytochrome c.

Example 5 Bcl-2 Inhibits Mitochondrial Release of Cytochrome C by BH3Peptides

Mitochondria bearing protective levels of anti-apoptotic BCL-2 do notrelease cytochrome c following treatment with 25 ng tBID in vitro,apparently because tBID is bound and sequestered by BCL-2 in stablecomplexes that prevent tBID from activating BAK (Cheng et al., 2001).Similarly, mitochondria with overexpressed BCL-2 proved resistant to 10μM BIDBH3, 1 μM BIMBH3, as well as 30 nM myrBID failing to releasecytochrome c (FIG. 5A) Furthermore, the presence of BCL-2 is coordinatewith the loss of BAK oligomerization following exposure to BH3 peptide,suggesting that BCL-2 inhibits upstream of BAK activation (FIG. 5B).These findings support a model wherein a major component of BCL-2's rolein inactivating tBID is to specifically sequester the BH3 domain, thuspreventing BH3 itself from activating multidomain pro-apoptotic members.

Example 6 BADBH3 Binds Bcl-2 and Restores Cytochrome C Release by BID

BH3 peptides were tested which lack the intrinsic ability to activateBAK and cause cytochrome c release for their capacity to interfere withthe anti-apoptotic protection by BCL-2. This subset of BH3 peptidesmight occupy the hydrophobic pocket of BCL-2 and consequently displaceproapoptotic BIDBH3 or BIMBH3 peptides. The BADBH3 peptide mostprominently demonstrates the capacity to overcome BCL-2 protection ofmitochondria treated with a subliminal concentration of myrBID (30 nM),while BIKBH3 shows significant, but lesser, potency (FIG. 6A). Theremaining BH3 peptides derived from NOXA and BCL-X_(L) did notdemonstrate the capacity to overcome BCL-2 protection (FIG. 6A). Sinceeven 100 μM BADBH3 in and of itself cannot activate BAK or releasecytochrome c, this suggests that BADBH3 sensitizes mitochondria toBIDBH3 or BIMBH3 by successfully competing with these peptides forbinding to BCL-2. At 100 μM, BADBH3 was able to restore the cytochrome crelease of BCL-2 overexpressing mitochondria in a dose-response fashionto BIDBH3 (FIG. 6B) and BIMBH3 (FIG. 6C) to levels observed for wtmitochondria. An increase in the sensitivity of wt mitochondria treatedwith BADBH3 was observed. This would be expected, as the source of wtmitochondria, FL5.12 cells, express some murine BCL-2. It was noted thatthe restoration of cytochrome c release by BADBH3 was accompanied byrestoration of BAK oligomerization.

Whether eliminating BCL-2 protection by BADBH3 would enable the morephysiologic ligand, myristoylated BID complex (p7/myrp15BID) was tested.Addition of 200 μM BADBH3 to BCL-2 overexpressing mitochondria markedlyrestores their sensitivity to even 1 nM myrBID. These BADBH3 treatedmitochondria are more sensitive than wt mitochondria, probablyreflecting the capacity of BADBH3 to inhibit the endogenous murine BCL-2and BCL-X_(L) resident on the mitochondria (FIG. 6D). The dose responserange of BADBH3 was examined, revealing it had measurable activity atconcentrations as low as 1 μM in inhibiting BCL-2 (FIG. 6E) and enablingcytochrome c release by myrBID. At 100 μM, BIKBH3 can also restorenear-total cytochrome c release to mitochondria over-expressing BCL-2,demonstrating a mechanism of action like BADBH3, albeit at higherconcentrations (FIG. 6E). This reveals that short BADBH3 and BIKBH3peptides can effectively compete with the natural myrBID protein forbinding BCL-2 thus abrogating BCL-2's antiapoptotic effect and enablingmyrBID induced cytochrome c release.

Example 7 BADBH3 Displaces BIDBH3 from BCL-2 by FluorescencePolarization Analysis

To directly test whether BADBH3 could displace BIDBH3 from BCL-2 weutilized fluorescence polarization analysis. BADBH3 peptide boundfull-length BCL-2 with approximately 5-fold greater affinity than BIDBH3peptide (average of 41 vs. 220 nM, Table 1, FIG. 7A). Moreover, BADBH3can efficiently displace pre-bound BIDBH3 peptide from BCL-2 (FIG. 7B).However, to compete pre-bound BIDBH3, an excess of BADBH3 is required,despite the 5-fold greater affinity of BADBH3 for BCL-2 in solution.This finding suggests a conformational change takes place in eitherBCL-2 and/or a BH3 peptide upon binding. In contrast, BIDBH3 does noteffectively displace BADBH3 from BCL-2. Testing the remaining peptidesreveals that those peptides which cause cytochrome c release bythemselves (BIDBH3 and BIMBH3) or those which enable cytochrome crelease by counteracting BCL-2 (BADBH3 and BIKBH3) all bind to BCL-2with affinities in the 50-500 nM range. BADBH3 and BIKBH3 demonstratethe ability to displace BIDBH3 from the BCL-2 protein (Table 1). Theremaining peptides (NOXABH3, NOXABBH3, BCLXBH3, BIDBH3mut) which wereunable to overcome BCL-2 inhibition did not bind detectably to BCL-2 ordisplace BIDBH3 from BCL-2 (Table 1). These results are consistent withthe capacity of “sensitizing” BH3 domains (e.g. BADBH3 or BIKBH3) todisplace “activating” BH3 domains (e.g. BIDBH3 or BIMBH3) from thepocket of anti-apoptotic BCL-2. Once free, “activating” BH3 domains bythis model would trigger BAK oligomerization with subsequent cytochromec release (FIG. 7C).

Whether apoptosis of cancer cells could be triggered by transduction ofsuch BH3 peptides was explored. Prior studies utilizing internalizationmoieties including decanoic acid, antennepedia (ANT) or HIV Tat havenoted confounding issues of cellular and mitochondrial toxicity. Forexample, when various BH3 domains were linked to a Tat 11-mer both wtand mutant transduced peptides rapidly killed cells. Moreover, manyconjugates did not appear to work through the genetic pathway as theydisplayed no inhibition by BCL-2 and readily killed Bax, Bak doublydeficient cells (not shown). Linking a polyarginine (8 aa) stretch tothe BH3 peptides appears more promising. Polyarginine tags have beenshown to facilitate the transport of peptides across the plasma membrane(Rothbard et al., 2000), r8BIDBH3 was capable of killing Jurkat leukemiccells, whereas r8BIDBH3mut was ineffective. Moreover, the addition ofnon-toxic 10YM r8BADBH3 was able to sensitize Jurkat cells to subliminalconcentrations (10 μM) of r8BIDBH3 (FIG. 8). Both r8BIDBH3 and r8BADBH3failed to kill Bax, Bak doubly-deficient cells. Thus, this appears toprovide an initial proof of concept experiment that “sensitizing” and“activating” BH3 domains will also synergize in vivo to initiateapoptosis of cancer cells.

Example 8

The use of synthetic peptides coupled with genetically definedmitochondria indicate that the BH3 peptide domain itself, excised fromthe context of an entire BH3-only molecule, can function as a specificdeath ligand. The activity of BH3 peptides supports a ligand/receptormodel in which “BID-like” BH3 domains are sufficient to triggerallosteric conformational activation of BAX, BAK, their respectivereceptors. Activation by BIDBH3 peptide was qualitativelyindistinguishable from the myrBID protein in that either requires BAK,results in BAK oligomerization followed by cytochrome c release, and canbe bound and sequestered by BCL-2 with resultant protection of BAK. Thesynthetic peptides also indicate that BH3 regions are true domainsrather than merely conserved sequence motifs, as the peptide domainitself has inherent functional activity. Comparison of variousca-helical peptides from BH3-only proteins reveals evidence for 2functional classes of BH3 domains. BID-like domains “activate”multidomain proapoptotic BAX, BAK. Whereas, BAD-like domains “sensitize”mitochondria for apoptosis by occupying the pocket of anti-apoptoticBCL-2. The latter displace BID-like domains which even at subliminallevels can now initiate cytochrome c release. This predicts thattherapeutics which mimic a BH3 domain, whether they be peptidomimeticsor small molecules, will be assignable to these functional classes andshould be classified utilizing the genetic and molecular reagentsdefined here.

From a therapeutic vantage point, BAD-like “sensitizing” BH3 mimeticswould possess several attractive characteristics. They might bepredicted to reset susceptibility of cells protected by BCL-2 orBCLX_(L), but would require a second apoptotic signal to initiate an“activating” BH3-only protein. This implies that as a single agent,“sensitizing” mimetics might prove non-toxic, especially to normalcells. The need for a second signal provides the opportunity to utilizecancer cell selective pathways that could also spare normal cells.

Evidence here for a “sensitizing” subset of BAD-like BH3 peptidesprovides an explanation for previous, apparent discrepancies concerningthe mechanism of action of these proteins. Most BH3-only intact proteinsincluding BAD, NOXA and BIK display a marked binding preference foranti-apoptotic members BCL-2, BCL-X_(L) in interaction assays of yeasttwo-hybrid, pull down, or co-immunoprecipitation from detergentsolubilized lysates (Boyd et al., 1995; Oda et al., 2000; Yang et al.,1995). Moreover, mutational analysis suggested that only when BAD wasable to bind anti-apoptotic BCL-X_(L) was it capable of promoting death(Kelekar and Thompson, 1998). Yet, BAD, NOXA and BIK all require themultidomain proapoptotic BAX, BAK proteins to kill as evidenced in Bax,Bak-doubly deficient cells (Cheng et al., 2001; Zong et al., 2001). Theability of BAD-like BH3 peptides to mediate a displacement reaction fromthe anti-apoptotic BCL-2 pocket provides a mechanism of action thatwould accommodate all observations. The cooperating protein displacedfrom anti-apoptotic pockets within intact cells would include, but notbe restricted to, BID-like “activating” BH3-only members. While helpingto resolve this issue, the analysis of the BIMBH3 peptide provedprovocative. Prior interaction assays indicate that the intact BIMprotein displays preferential binding to anti-apoptotic BCL-2, BCL-X_(L)over proapoptotic BAX or BAK. Previous reports testing the capacity ofintact BIM protein to release cytochrome c from mitochondria gavediffering results (Li et al., 2001; Terradillos et al., 2002). Here, theisolated BIM BH3 domain when removed from the context of the entireprotein scored as BID-like, capable of activating BAX, BAK. Severalpotential explanations can be envisioned. It is possible that thecritical α-helical face of the BH3 domain that recognizes BAX, BAK maynot be exposed in the intact BIM protein. Alternatively, it is alsoconceivable that the standard protein interaction assays used to measurebinding may not reflect all of the conformational states that a nativeBIM molecule undergoes during cell death in-vivo.

The mechanistic pathway to cytochrome c release for BIDBH3 peptideappears similar to native myrBID complex, yet the efficiency oftriggering varies greatly. Near total release of cytochrome c frommitochondria requires but 10 nM myrBID complex, but 10 μM BIDBH3peptide. Myristoylation increases the efficiency of BID targeting tomitochondria and could conceivably help focus its location on the outermitochondrial membrane (Lutter et al., 2001; Zha et al., 2000). It isalso possible that an integrated myrp15BID protein may more effectivelypresent the BH3 domain to the BAK pocket. Of note the sources ofmitochondria vary in their response to individual BH3 domains. BIDBH3 ismore potent than BIMBH3 for liver mitochondria, whereas BIMBH3 is moreeffective on the FL5.12 mitochondria. This may reflect the presence ofBAX on FL5.12 but not liver mitochondria. The efficiency ofoligomerization (FIG. 4) supports a preference of BIDBH3 for BAK andBIMBH3 for BAX. An hypothesis that BIMBH3 prefers BAX would beconsistent with the finding that BIM functions upstream of BAX inneuronal cell death following NGF deprivation (Putcha et al., 2001). Thebinding affinity of individual BH3 domains for BCL-2 members variesconsiderably (Sattler et al., 1997) (FIG. 7) providing a measurement forselectivity. Assessment of BH3 peptides by circular dichroism indicatesthat α-helical content is not the sole determinant of differentialbinding affinity, nor for the ability to induce BAX, BAK oligomerizationand cytochrome c release. The specificity noted suggests a model inwhich distinct BH3 domains have select multidomain partners whichprovides a rationale for the large number of both BH3-only andmultidomain antiapoptotic members.

Whether the BH3 domains of multidomain members can initiate apoptosis isless certain. The BH3 domain isolated from BCL-X_(L) studied here showedno activity, while BH3 peptides from BAX have generated mixed results.Addition of a BH3 peptide from BAK to a Xenopus cell free system inducedrelease of cytochrome c and caspase activity, although the site ofaction was unknown (Cosulich et al., 1997). Addition of BAXBH3 tomammalian mitochondria has been reported to release cytochrome c withoutinducing permeability transition (Polster et al., 2001), consistent withthe mechanistic pathway dissected here; whereas, others report BAXBH3peptides which do induce permeability transition and loss oftransmembrane potential as an explanation for cytochrome c release(Narita et al., 1998). This may be inherent to α-helices themselves orto hybrid proteins which can damage organelle membranes.

A substantial challenge for the future is to effectively transduce BH3peptides or BH3 peptidomimetics into cells and assure that the inductionof apoptosis is through the genetic pathway. Several studies (Holingeret al., 1999; Wang et al., 2000) including the initial polyargininetransduction approach presented here suggest this warrants furtherefforts. However, caution exists as a number of amphipathic α-helicalpeptides, especially if they are cationic, can be attracted tonegatively charged membranes, including mitochondrial membranes wherethey can non-specifically disrupt the lipid matrix and membrane barrierfunction (Ellerby et al., 1999; Matsuzaki, 2001; Westerhoff et al.,1989). Others utilizing ANT-BH3BAD found toxicity was independent of theBCL-2 pathway and also killed yeast, which tolerate expression of theBH3-only proteins (Schimmer et al., 2001; Vieira et al., 2002).Alternative methods of internalization including receptor mediatedpathways should also be considered for BH3 peptidomimetics. The workhere provides a proof of concept that BH3 mimetics can be designed whichinitiate apoptosis correctly, at definable points in the geneticpathway. Moreover, it provides a paradigm and reagents to dissect themechanism of action of future BH3 mimetics.

Example 9 Screening for BH3-Mimetics Using a Mitochodrial Assay

BH3-mimetics, e.g., BAD-like or BID-like, are screened using amitochondrial assay. (See, FIG. 9) Mitochondria from livers of mice thatare either wild-type or Bak−/− are isolated. Isolated mitochondria arecontacted with a test compound and cytochrome c release is determined.No cytochrome c release in either wt or Bak−/− mitochondria indicatesthe test compound is a “sensitizing”, BAD-like mimetic. To further testthis potential “sensitizing” BAD-like mimetic is added to subliminalconcentration of a BID-like mimetic myrBID itself to determine ifcytochrome c release is enhanced from BAK wt mitochondria. A BAD likemimetic is useful for example as a specific BCL-2/BCL-X_(L) inhibitor. Acompound that induces cytochrome c release in wt but not Bak−/−mitochondria indicates the compound is an “activating” BID-like mimetic.BADBH3 and myrBID are used as controls for sensitizing and activatingcompounds respectively.

Candidate sensitizing compounds are further characterized by isolatingmitochondria from either wt or over expressing BCL-2 FL5.12 cells.Sensitizer compound do not induce cytochrome c release from wt Fl5.12mitochondria, however sensitizer compounds do enable subliminalconcentrations of myrBID or a BID-like mimetic to release cytochrome cfrom BCL-2 protected mitochondria.

A candidate sensitizing compound is further tested for its ability tokill BCL-2 dependent rather than BCL-2 independent cancer cells inculture.

Example 10 Production and Characterization of Bcl-2 Conditional KnockinMouse

Using standard recombinant DNA technology, the tet-BCL-2 allele wastargeted to the DNA methyltransferase 1 locus (FIG. 10). By crossing thenew mouse line with the MMTV-tTA line (Jackson Labs) mice were generatedwhich conditionally overexpress BCL-2 in epithelial cells and inB-lineage lymphocytes. Administration of doxycycline successfullysuppresses BCL-2 overexpression in these animals. When these mice arecrossed with mice expressing myc in the B-lymphocyte lineage (Eμ-mycmice, Jackson Labs) mice which uniformly have B-cell lymphoblasticleukemia were obtained.

To examine the effect of removing BCL-2 expression form the leukemiacells, the effects of doxycycline treatment in a cohort of 28 mice wereexamined. By genotype, each of the 28 mice contained the tet-BCL-2, theMMTV-tTA, and the Eμ-myc alleles. Phenotypically, each were found tohave leukemia by blood smear, and a WBC in excess of 100,000/μl. At 4-5weeks of age, half were treated with doxycycline (500 μg/ml) in drinkingwater, and half were left untreated. The WBC was measured byhemocytometer using 2 μl blood from tail which was lysed with 0.3%saponin and stained with Hoechst 33258. As shown in FIG. 11 that theloss of BCL-2 expression induced by doxycycline treatment induces adramatic, 1-2 log decrease in WBC and a remission of the leukemia. Thesedata provide strong support for the requirement of BCL-2 for tumormaintenance in this murine cancer model, and validates this model forfuture in vivo testing of candidate BCL-2 inhibitors.

Example 11 Propagation of a Cell Lines Derived from the Bcl-2Conditional Knockin Mouse

A culture a cell line from the bone marrow of a triply transgenic,leukemic mouse was propagated. When BCL-2 transgene expression iseliminated by doxycycline treatment, the cell line dies. This BCL-2dependent cancer cell line is useful for cellular testing of candidateBCL-2 inhibitors.

Furthermore, as a control for specificity of a putative BCL-2antagonist, a cell line from B-cell malignancies which are not dependenton BCL-2 expression were generated. The tumors arose in triplytransgenic mice that had not been maintained on doxycycline to abolishBCL-2 transgene expression. Cell from these mice were grown in culturein the absence of doxycycline. Turning off BCL-2 expression byadministration of doxycycline did not induce cell death. These resultsdemonstrate that these cell lines are useful in conjunction with theBCL-2 dependent cell line described above to assess the specificity ofaction of a putative BCL-2 antagonist. A specific BCL-2 antagonist is acompound that kills the BCL-2 dependent cell line but does not kill theBCL-2 independent cell line.

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Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A composition comprising an isolated BH3 peptideof less than or equal to 50 amino acids in length, and a mitochondrion.2. The composition of claim 1, wherein the BH3 peptide is apro-apoptotic BID-like BH3 peptide.
 3. The composition of claim 2,wherein the BID-like BH3 peptide includes an amino acid sequenceselected from SEQ ID NOs: 1, 2, and
 10. 4. The composition of claim 1,wherein the BH3 peptide is an anti-anti-apoptotic BAD-like BH3 peptide.5. The composition of claim 4, wherein the BAD-like BH3 peptide includesan amino acid sequence selected from SEQ ID NOs: 3-7 and
 11. 6. Thecomposition of claim 1, wherein the mitochondrion is isolated from acell.
 7. The composition of claim 1, wherein the BH3 peptide is lessthan or equal to 35 amino acids in length.
 8. The composition of claim1, wherein the BH3 peptide is less than or equal to 25 amino acids inlength.
 9. The composition of claim 1, wherein the BH3 peptide is lessthan or equal to 15 amino acids in length.
 10. The composition of claim1, wherein the isolated BH3 peptide is in a complex with a BCL-2 orBCL-XL protein resident on the mitochondrion.
 11. The composition ofclaim 1, wherein the isolated BH3 peptide is in a complex with a MCL-1protein resident on the mitochondrion.
 12. A composition comprising amitochondrion contacted with an isolated BH3 peptide of less than orequal to 50 amino acids in length.
 13. The composition of claim 12,wherein the BH3 peptide is a pro-apoptotic BID-like BH3 peptide.
 14. Thecomposition of claim 13, wherein the BID-like BH3 peptide includes anamino acid sequence selected from SEQ ID NOs: 1, 2, and
 10. 15. Thecomposition of claim 12, wherein the BH3 peptide is ananti-anti-apoptotic BAD-like BH3 peptide.
 16. The composition of claim15, wherein the BAD-like BH3 peptide includes an amino acid sequenceselected from SEQ ID NOs: 3-7 and
 11. 17. The composition of claim 12,wherein the mitochondrion is isolated from a cell.
 18. The compositionof claim 12, wherein the BH3 peptide is less than or equal to 35 aminoacids in length.
 19. The composition of claim 12, wherein the BH3peptide is less than or equal to 25 amino acids in length.
 20. Thecomposition of claim 12, wherein the BH3 peptide is less than or equalto 15 amino acids in length.
 21. The composition of claim 12, whereinthe isolated BH3 peptide is in a complex with a BCL-2 or BCL-XL proteinresident on the mitochondrion.
 22. The composition of claim 12, whereinthe isolated BH3 peptide is in a complex with a MCL-1 protein residenton the mitochondrion.